Targeting CD19 in diffuse large B‐cell lymphoma: An expert opinion paper

Abstract The ubiquitous, early‐stage expression, efficient internalization, limited off‐target effects, and high disease specificity of CD19 make it an attractive therapeutic target. Currently available anti‐CD19 therapies have demonstrated particular promise in patients with relapsed or refractory B‐cell non‐Hodgkin lymphoma. Selection of the most appropriate treatment strategy should be based on individual patient characteristics and the goal of therapy. However, evidence and knowledge about the sequencing of anti‐CD19 therapies are limited. Here, we review the current evidence for CD19 as a target in diffuse large B‐cell lymphoma and consider approaches to the use of anti‐CD19 therapy.


| INTRODUCTION
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL), constituting around 25%-40% of cases worldwide. 1,2 The annual incidence of DLBCL is three to four per 100,000 persons in Europe and 7 per 100,000 persons in the United States, 3,4 with diagnosed incident cases predicted to increase steadily over the coming years. 5 The introduction of the anti-CD20 monoclonal antibody rituximab in 1997 revolutionized the treatment of DLBCL. 6 Rituximab-mediated cell death is thought to occur through several different mechanisms including natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC), antibodydependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and direct antitumor effects via apoptosis or other cell death pathways. 7,8 Rituximab given in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) was approved in 2006 as first-line treatment for patients with DLBCL and remains the standard therapy for previously untreated patients, achieving complete and sustained remission in approximately 60% of this population. [9][10][11] However, around 30%-40% of patients relapse following an initial response to therapy or are unable to achieve remission with first-line treatment. 1 Among patients with relapsed/refractory (R/R) DLBCL, 30%-40% have a response to salvage chemotherapy and up to 50% of these patients subsequently undergo autologous stem-cell transplantation (ASCT), with around half ultimately relapsing after transplantation. 1,3 Patients with R/R DLBCL currently have few treatment options, and outcomes are poor due to a lack of durable response to salvage chemotherapy. 12 Rituximab-containing regimens are frequently used, despite limited evidence supporting their use. 2 Various mechanisms of resistance to rituximab have been proposed, 13 with CD20-negative change after rituximab-containing therapy believed to be one of the main resistance mechanisms in B-cell NHLs. This is thought to occur as a result of epigenetic downregulation of CD20 messenger ribonucleic acid expression, homozygous deletion of the CD20 gene, and selection of CD20negative clones. 14 Research suggests that many patients with DLBCL may have a low peripheral blood NK cell count, and this may be associated with poorer outcomes with anti-CD20-based therapy, including shorter progression-free survival (PFS), versus patients who have higher NK cell counts. 15 A considerable focus has been placed on improving outcomes of first-line treatment and increasing cure rates in newly diagnosed DLBCL, 16 although there is limited evidence demonstrating a consistent, clear benefit compared with R-CHOP. Preliminary results from the POLARIX trial suggest that treatment with polatuzumab vedotin in combination with R-CHOP improves PFS compared with standard of care R-CHOP in patients with DLBCL, but further data are awaited to confirm this. 17 Efforts to improve outcomes for patients with R/R DLBCL are also ongoing through the development of novel agents with new targets, including the Bcell surface antigens CD19, CD22, CD37, and CD79B. 18 Among these, CD19 shows particular promise, since it is constantly and strongly expressed in the vast majority of B-cell lymphoproliferative diseases, is highly disease-specific, and has a limited number of off-target effects. 19,20 In this paper, we examine the role of CD19 in the pathophysiology of DLBCL, review the evidence for CD19 as a target in DLBCL, and consider approaches to the use of anti-CD19 therapy.

| NATURAL ROLE OF CD19 AND ROLE IN B-LYMPHOMAGENESIS
CD19 is a type 1 transmembrane glycoprotein that belongs to the immunoglobulin (Ig) superfamily. 21,22 It consists of a single transmembrane domain, a cytoplasmic C-terminus, and extracellular Nterminus ( Figure 1). 22  is expressed ubiquitously on the surface of B cells from the early to mature stages of development, 31 and is subsequently downmodulated at the plasma cell stage. 23,32 CD19 is not shed from the surface of B cells into the circulation. 33 The expression profile of CD19 is broader than that of CD20 34 and CD19 is expressed at an earlier pre-B stage than CD20. 35 In normal cells, CD19 plays a key role in many B-cell functions, including development and differentiation, proliferation, and signaling. 23 Tafasitamab has shown potent activity in preclinical studies involving lymphoma and leukemia models, including 100-1000-fold enhanced ADCC relative to an anti-CD19 IgG1 analog in vitro. 41,42 Tafasitamab has demonstrated single-agent activity in the setting of R/R DLBCL and other B-cell malignancies, with some patients achieving durable responses. [44][45][46][47] Preclinical evidence also suggests that tafasitamab acts synergistically in combination with lenalidomide. 42 Lenalidomide has direct antineoplastic activity and indirect effects mediated via various immune cells in the tumor microenvironment. 48 In particular, lenalidomide stimulates NK-cell proliferation and activation, thus enhancing NK-cell-mediated ADCC by tafasitamab in vitro. 42 The phase two L-MIND trial (NCT02399085) investigated the combination of tafasitamab plus lenalidomide followed by tafasitamab monotherapy in 80 patients with DLBCL who relapsed or were refractory after one to three systemic regimens (including at least one CD20-targeting regimen), and who were ineligible for ASCT. Exclusion criteria included primary refractory DLBCL, defined as no response to, or progression during or within 6 months of, frontline therapy. Fifteen patients (19%) with primary refractory disease were included in the study. Prior to a protocol amendment, patients who relapsed within 3 months of a prior anti-CD20-containing regimen were defined as primary refractory and were excluded from L-MIND.
Patients who relapsed or progressed between 3 and 6 months after receiving frontline therapy recruited before the amendment were considered as primary refractory but were included. In total, 42.0% The 12-month DOR was similar regardless of refractory status to last therapy, although the 12-month PFS and OS were lower compared with non-refractory patients. 52 Responses were observed in two patients who had double-or triple-hit lymphoma. 45 In a patient-level

| Anti-CD19 bispecific T-cell engagers: blinatumomab
Strategies to target CD19 focusing on T cell recruitment led to the development of blinatumomab, a CD19xCD3 bispecific T-cell engager that directs cytotoxic T-cells to lyze CD19-expressing B cells. [60][61][62][63] Blinatumomab exhibits linear pharmacokinetics and due to its short elimination half-life (t 1/2 1-2 h), must be administered as a 24-h continuous intravenous infusion. 64 Blinatumomab has demonstrated activity in three phase 1/2 trials in heavily pre-treated patients with aggressive R/R DLBCL, with CR rates ranging from 19% to 22%. 62,65 In a pooled analysis of these studies, the median duration of CR and

| Anti-CD19 antibody-drug conjugates: loncastuximab tesirine
The characteristics of CD19 (i.e., rapid internalization kinetics, broader expression profile than CD20, not shed into the circulation) make it a better target for antibody-drug conjugates (ADCs) than CD20. 34 Data from preclinical studies suggest that adding rituximab to an anti-CD19 pyrrolobenzodiazepine ADC prolongs tumor control, providing a rationale for combining loncastuximab tesirine with rituximab as a treatment for R/R DLBCL. 75 The phase three, randomized, open-label, two-part LOTIS-5 trial (NCT04384484) is investigating the efficacy of loncastuximab tesirine with rituximab versus rituximab/gemcitabine/oxaliplatin as standard immunochemotherapy in patients with R/R DLBCL. 75,76 In a pooled safety analysis of loncastuximab tesirine, grade ≥3 neutropenia, thrombocytopenia and anemia occurred in 32.1%, 20.0% and 12.6% of patients with R/R DLBCL, respectively. Most of these events were manageable with dose delays, and did not require dose reduction or discontinuation of treatment. 77 Liver enzyme elevations are also common with loncastuximab tesirine, which are thought to be due to the pyrrolobenzodiazepine dimer warhead part of the ADC. 78 Loncastuximab tesirine received FDA approval for use in patients with R/R DLBCL who have received at least two prior systemic therapies. 79 Relapse after CAR T-cell therapy is common, with around half of relapses occurring within the first month in R/R DLBCL. 104 Risk factors for early progression are two or more extranodal sites, increased C-reactive protein level, and high total metabolic tumor volume at the time of treatment. 104 In ZUMA-1, CD19-negative relapse occurred in around 30% of patients following axi-cel therapy. 105 Though the mechanisms responsible for this are not yet fully clear, it is thought to be due to the emergence of tumor cells with low or no CD19-antigen expression within the context of targeted removal of antigen-positive tumor cells. 105 Local resistance mechanisms within tumors may also play a role in relapse after CAR T-cell therapy. Lesions at high risk for local failure include high metabolic activity, diameter ≥5 cm, and extranodal disease. It is hypothesized that resistance to CAR T-cells may occur at the individual lesion level,

| Anti-CD19 chimeric antigen receptor T-cell therapy
given that a discordant response to therapy is frequently observed, with some lesions remaining in remission and others progressing. 100

| Other anti-CD19 therapies in development with potential for use in R/R diffuse large B-cell lymphoma
Inebilizumab is a humanized, anti-CD19 monoclonal antibody that targets and depletes CD19-expressing B cells via ADCC. 106 In a phase one dose-escalation study that included six patients with R/R DLBCL, the ORR was 50%; one patient achieved a CR and two patients achieved a PR. 107 Bicistronic CAR constructs have been engineered using a single vector that encodes two different CARs on the same cell, allowing dual targeting of CD19 and CD20, thereby overcoming the loss of one antigen. 108

| SEQUENTIAL USE OF ANTI-CD19 THERAPY
The recent approval of novel, CD19-directed therapies for DLBCL presents a challenge in determining the optimal sequence and duration of treatments for an individual patient. 1 Level of CD19 expression is not currently used as a decision tool for anti-CD19 therapy since CD19 expression is highly conserved, with normal to high levels of expression maintained on nearly 90% of B-cell lymphomas. 22 The absence of CD19 expression may be of relevance for treatment decisions following CAR T-cell therapy, as discussed further below.
With CAR T-cell therapy, the intent is curative in the setting of refractory disease. With other anti-CD19 targeted therapies, the goal is to prolong remission and extend survival, with acceptable treatment tolerability. Although CAR T-cell therapies can achieve durable responses, they are not currently approved for use in the second-line setting and various barriers prevent their widespread uptake, meaning only selected patients will benefit. 109 including at least one CD20-targeting regimen, adequate organ function, ECOG performance status of 0-2, measurable disease). 45 Although the goal of therapy differs with these two approaches, the 43.9-month median DOR with tafasitamab plus lenalidomide in L-MIND is similar to that which can be achieved with CAR T-cells. 47 In a long-term follow-up of patients with DLBCL treated with anti-CD19 CAR-T cells, 48% of treatments resulted in a DOR of over 3 years 119 However, it is important to note that the characteristics of patients in L-MIND differed from those in trials of CAR T-cell therapy.

| Does previous use of anti-CD19 therapy preclude the use of CAR-T cells or subsequent activity of other anti-CD19 therapy?
It is not yet known whether the CD19 antigen can be targeted with a different anti-CD19 therapy after disease progression following a previous CD19-directed therapy. There are concerns regarding antigen masking and the potential for selection pressure of the prior therapy, which could lead to CD19 antigen escape. 120  -511 which are encoded by exon 2 and centered around residue R144 (exon 2 encodes a portion of the extracellular domain of the transmembrane protein). 41,[125][126][127] Phase one data from 14 patients with chronic lymphocytic leukemia showed that CD19 expression levels were maintained on the surface of CD24-positive B cells following tafasitamab treatment. 128 Preclinical evidence indicates that exposure to tafasitamab does not impair subsequent anti-CD19 CAR T-cell binding in vitro. Various cell lines including DLBCL were incubated with tafasitamab to saturate the CD19 antigens. CD19 saturation did not affect CAR T-cell effector functions such as cytokine production, degranulation, proliferation, or antigen-specific killing. 129 Evidence suggests that low or undetectable CD19 expression levels may be adequate for effective anti-CD19 CAR T-cell therapy.
In an analysis of CD19 expression in pre-infusion biopsies taken from patients in the JULIET trial of tisa-cel, similar response rates were observed irrespective of the level of CD19 expression. 84 CD19 status prior to CAR T-cell therapy is not assessed in some countries, while in others CD19-negative status is an exclusion criterion. Immunohistochemistry is most commonly used for CD19 analysis, but may not be a reliable method of assessment. Different CD19 antibodies have distinct epitopes yet most laboratories use only one CD19 antibody for immunohistochemistry, which could yield false negative results. Furthermore, unlike flow cytometry, CD19 staining by immunohistochemistry is not standardized and is highly variable between laboratories.

| Use of anti-CD19 therapies as bridging therapy
The optimal bridging therapy strategy between leukapheresis and CAR T-cell therapy remains to be determined. Currently, the goal is to minimize total metabolic tumor volumes with the use of a short bridge during CAR T-cell manufacturing. However, approaches are likely to change as new data become available.
The use of bridging therapy was an exclusion criterion in the ZUMA-1 trial of axi-cel but was allowed in the JULIET trial of tisa-cel; however, not all patients received it. While evidence is lacking, we currently advise against the use of anti-CD19 therapy before potentially curative CAR T-cell therapy, due to the risk of downregulating the antigen.

| Use of anti-CD19 therapy following CAR T-cell therapy
It is estimated that 30%-60% of patients will progress after CAR Tcell therapy, with poor outcomes. 130 Current options for these patients are entry into a clinical trial (e.g., of a bispecific antibody), tafasitamab plus lenalidomide or polatuzumab vedotin plus bendamustine and rituximab (where approved), or palliative oral chemotherapy. If CAR T-cell therapy is approved in the second-line setting, it might be estimated that around 35% of patients would be 'cured' and 65% would be available for salvage therapy; of this latter group, 30% of patients will lose the CD19 antigen and the remainder will have CD19 expression detectable by immunohistochemistry and could be considered for anti-CD19 immunotherapy.

| CONCLUSION
The early-stage expression and efficient internalization of CD19 and as an advisor for AbbVie, Gentili, Gilead, Janssen-Cilag, Novartis, and Roche.

DATA AVAILABILITY STATEMENT
The author has provided the required Data Availability Statement, and if applicable, included functional and accurate links to said data therein.

DATA SHARING
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.